There are a variety of dyes available for the detection of proteins and peptides on gels, particularly polyacrylamide gels, after the proteins or peptides have been separated by electrophoresis, chromatography, or other method. However, there is a continuing need for increasingly sensitive agents which may accurately detect presence of proteins in smaller amounts and in particular which allow quantitative determination of the protein. Thus, there is a continuing need for dyes which are accurate using diminished protein loads while retaining versatility in detecting a wide variety of proteins and peptides.
One of the more sensitive detecting methods employs the use of staining procedures based upon the precipitation of silver on the protein, the so-called silver-staining method. There are different disadvantages however with the silver-staining method, one of which being that the color of silver-stained spots may fade and/or change color with time. The silver-staining proceedure itself is rather time consuming and requires many steps. Furthermore, because the detection threshold and slope of the mass/grain intensity relationship utilizing silver stains differs for various proteins and with various silver stains, reliable results may not be consistently obtained. Probably most importantly, the silver-staining technique is not adequate for quantitative determination of the protein. See Zapolski, et al., Electrophoresis, 5, 354-357 (1984).
The class of dyes known as the Coomassie.RTM. dyes, in particular Coomassie Blue R-250, although not quite as sensitive as silver stains, has been a widely used dye due to its versatility, ease of use, and its adaptability for quantitative determination of proteins. However, the usefulness of Coomassie dye is still limited by its sensitivity.
In a search for more sensitive dyes, Zapolski, et al., supra, made a variety of .sup.35 S-labeled stains for the detection of proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels. However, the study was not made for quantitative use of the dyes and the authors determined that the .sup.35 S-labeled Coomassie Blue R-250 was not a promising dye for this purpose. Zapolski, et al., Analytical Biochemistry, 123, 325-328 (1982) also studied the .sup.59 Fe-labeled ferrous bathophenanthroline sulfonate (BPS) as a protein-staining dye. However, use for quantitative work was not determined nor was its versatility for use with a variety of proteins.
Autoradiography has also been utilized to detect proteins on gels. However, this technique requires the labeling of the proteins to be detected prior to their application onto the gel. However, many types of protein samples, cannot be readily radiolabeled, such as human proteins, and therefore are not amenable to autoradiography.
There is therefore a continuing need to develop materials for detection of proteins on gels which are improved in one or more of the following aspects: ease of use, safety in handling, sensitivity, versatility, applicability for quantitative work.
It is therefore an object of the present invention to provide an improved method for detecting proteins on gels, both in qualitative and quantitative applications, using dyes with enhanced sensitivity as well as versatility, ease and safety of use.
These and other objects will become apparent from the following description of the preferred embodiments and from the appended claims.